Cell cycle is a fundamental program that exists to precisely regulate mitotic fidelity and cell proliferation in uni- and multi-cellular organisms. The basic stages of the cell cycle are conserved from yeast to humans and include G1, S, G2, and M. Significant clinical, genetic and cell biologic evidence shows that disruption of cell cycle regulation results in aberrant cell proliferation and is central to carcinogenesis. For example, human tumor specimens frequently demonstrate an increased mitotic index, as shown by increased BrdU incorporation and PCNA expression. In addition, most oncogenes directly affect cell proliferation, acting as transmembrane receptors (ERBB, RET), membrane-bound (SRC, RAS) or cytoplasmic (ABL) signaling molecules, or transcription factors (MYC, JUN).
Many genes control the processes required for normal cell proliferation and when these genes are mutated, abnormal proliferation and tumor formation result. In humans, only a few genes in this complicated process have been characterized and a screening method of identifying genes specifically involved in cell cycle using a forward genetic approach would be advantageous.
Mice offer some advantages as a model organism for the study of cancer genes. Many homologues of the cloned human tumor suppressor genes have been mutated in the mouse [McClatchey, A., et al., Curr Opin Genet Develop, 8:304-310, 1998]. By obtaining strains carrying germline disruptions of these genes, both the heterozygous and homozygous phenotypes can be studied. Mice having heterozygous loss-of-function mutations represent models of humans with familial cancer syndromes and can serve as a model system for study of the progression of cancer. Additionally, the homozygous mutants can reveal developmental roles of these tumor suppressor genes. The generation of mouse strains with combinations of tumor suppressor gene mutations provides information about the genetic interactions in tumorigenesis. Transgenic mice expressing oncogenes provide information about the effects these genes have on proliferation and differentiation [Eva A., Semin Cell Bio, 3:137-45, 1992]. However, mice are not ideal animals for forward genetic studies to help to identify genes by their function as the number of mice needed for performing a genome-wide screen for recessive mutations is difficult and costly to maintain [Hrabe de Angelis M. et al., Mutat Res, 400:25-32, 1998].
Drosophila is another genetic model system for the study of cancer. The first mutant gene was identified as lethal (2) giant larvae gene (1(2)g1) and showed homology to a human gene [Mechler B. M, et al., EMBO J. 4:1551-57, 1985]. Genetic screens have identified mutations in over 50 genes in larval and adult stages [Watson K L, J. R., et al., Cell Sci Suppl, 18:19-33, 1994]. Many of these germ-line mutations cause embryonic lethality in homozygous animals, so screening for additional genes has been done in mosaic flies [Xu T., et al., Development, 121:1053-63, 1995]. Genes identified in this screen, such as LATS (large tumor suppressor), have proven to be relevant in mammals since knockout LATS-mice develop soft tissue sarcomas, ovarian tumors and pituitary dysfunction [St John, M. A., et al., Nat Genet, 21:182-186, 1999]. However, although Drosophila has revealed important genes involved in cancer, Drosophila tumor pathology does not closely resemble human tumors. Therefore, it would be an advantage to have a vertebrate forward genetic system to study cancer development that more closely resemble humans.
Fish have been utilized in laboratory carcinogenesis studies to evaluate the risk from environmental hazards [Couch, J., Toxicol Pathol, 24:602, 1996]. Zebrafish have been an integral part in these studies, and therefore much is known about carcinogen effects and tumor formation in this species. Zebrafish are known to develop numerous types of tumors, both naturally and through induction from genotoxic agents [Spitsbergen J. M., et al., Toxicol Pathol. 28:716-25, 2000; Khudoley, V. V., Natl. Cancer Inst. Monogr, 65:65-70, 1984]. Recently, transgenic zebrafish have been used for detecting mutations induced by particular compounds [Amanuma, K., et al., Nat Biotechnol, 18:62-65, 2000].